RUI: Mutagenesis Studies to Probe Function of the Large Extrinsic Loop of the CP-43 Apoprotein of Photosystem II

该项目描述了在光系统II （PSII）蛋白CP43的大外源性环区含有随机和定点病变的突变体的产生和特征。CP43是一种完整的类囊体蛋白，是高等植物、藻类和蓝藻的光合器官的组成部分。这些突变体将在蓝细菌Synechocystis 6803中构建，这是一种广泛使用的高等植物光合作用模式系统。CP43似乎是PSII复合体稳定组装所必需的，并且在光合作用的氧气演化中也起作用。这个项目的目标是确定CP43的大的、面向腔的外部环的功能。环的长片段的删除导致突变体不能进化氧气。因此，该区域可能与其他PSII蛋白和无机辅因子（锰、氯和钙）直接相互作用，这些辅因子是析氧所必需的。此外，CP43还作为叶绿素-a天线对PSII起作用。因此，大的外源性环可能与其他PSII天线蛋白或包含PSII反应中心的蛋白相互作用。突变将通过随机和寡核苷酸介导的突变引入编码CP43的psbC基因。突变将与密切相关的抗生素耐药性标记一起重新引入聚囊菌6803。假定的突变体将首先筛选抗生素耐药性和可能的光自养生长损失。随后将深入表征光合特性，包括测量稳态氧释放速率、电子传递能力、PSII多肽组成、锰和钙含量以及氯化物需求。光合系统II （PSII~ complex）是高等植物、藻类和蓝藻的光合器官的一部分。该络合物催化水的光氧化和塑醌的还原，氧作为PSII反应的副产物释放出来。CP43是PSII的蛋白质组分之一，似乎是PSII复合物稳定组装所必需的。它似乎也在光合作用的氧气进化和光能的收集中发挥作用。这种膜结合蛋白是不寻常的，因为它含有一个大的亲水性环，从它所嵌入的膜上伸出来。大外源性环中氨基酸短片段的缺失导致突变体不能进化出氧气。该项目的目标是确定参与CP43的大的、面向腔的外源性环的功能的特定氨基酸，以进一步验证该区域是正常PSII活性所必需的假设。这将通过1)在体外使用随机和位点特异性诱变技术将突变引入编码CP43的基因中，2)将这些突变基因交换为蓝绿藻Synechocystis 6803中的正常对应基因，3)评估产生的突变生物组装正常PSII复合物、进化氧气和进行电子传递的能力。* * * ? ?

9513795 Putnam-Evans This project describes the production and characterization of mutants containing random and site-directed lesions in the large extrinsic loop region of the Photosystem II (PSII) protein CP43. CP43 is an integral thylakoid protein and component of the photosynthetic apparatus of higher plants, algae and cyanobacteria. These mutants will be constructed in the cyanobacterium Synechocystis 6803, a widely used model system for higher plant photosynthesis. CP43 appears to be required for the stable assembly of the PSII complex, and also appears to play a role in photosynthetic oxygen evolution. The goal of this project is to determine the function of the large, lumenally-oriented extrinsic loop of CP43. Deletion of long segments of the loop results in mutants incapable of evolving oxygen. Therefore, it is probable that this region interacts directly with other PSII proteins and inorganic cofactors (manganese, chloride and calcium) that are necessary for oxygen evolution. In addition, CP43 functions as a chlorophyll-a antenna for PSII. The large extrinsic loop may thus interact with other PSII antenna proteins or proteins comprising the PSII reaction center. Mutations will be introduced into the psbC gene encoding CP43 by both random and oligonucleotide-mediated mutagenesis. Mutations will be re-introduced into Synechocystis 6803 along with a closely-linked antibiotic resistance marker. Putative mutants will be screened initially for antibiotic resistance and possible loss of photoautotrophic growth. This will be followed by an in-depth characterization of photosynthetic characteristics including measurements of steady state rates of oxygen evolution, electron transport capability, PSII polypeptide composition, manganese and calcium content and chloride requirement. %%% The Photosystem II (PSII~ complex forms part ofthe photosynthetic apparatus of higher plants, algae and cyanobacteria. This complex catalyzes the light-driven oxidation of water and the reduction of plastoquinon e. Oxygen is given off as a by-product of the PSII reaction. CP43 is one of the protein components of PSII and appears to be required for the stable assembly ofthe PSII complex. It also appears to play a role in both photosynthetic oxygen evolution and the harvesting of light energy. This membrane-bound protein is unusual in that it contains a large, hydrophilic loop which protrudes from the membrane in which is it embedded. Deletion of short stretches of amino acids in the large extrinsic loop result in mutants that cannot evolve oxygen. The goal of this project is to identify specific amino acids involved in the function of the large, lumenally-oriented extrinsic loop of CP43 to further test the hypothesis that this region is necessary for normal PSII activity. This will be accomplished by 1) introducing mutations into the gene encoding CP43 in vitro using the techniques of both random and site-specific mutagenesis, 2) exchanging these mutated genes for their normal counterparts in the blue-green algae Synechocystis 6803 and 3) evaluating the resulting mutant organisms for their ability to assemble a normal PSII complex, evolve oxygen and carry out electron transport. *** ??